Corynebacteriophage Betatox+ and a family of closely related corynephage carry the structural gene for diphtheria toxin. Portions of the tox gene have been cloned in Escherichia coli, and fragment A of toxin has been shown to be expressed from the tox promoter and exporter to the periplasmic compartment by the SecA apparatus. The specific aims of the present proposal are to continue the study of diphtheria tox regulation of expression through (i) a detailed analysis of the tox promoter as it functions both in Corynebacterium diphtheriae and in E. coli and (ii) a detailed analysis of the putative tox operator and the molecular cloning of the C. diphtheriae repressor. It is further proposed that our study of diphtheria toxin structural/functional domains be continued. Specifically, we will continue the study of the location and amino acid sequence of the toxin eukaryotic cell receptor binding domain, as well as the putative processing domain in fragment B which appears to be absolutely necessary for the membrane translocation of fragment A into the eukaryotic cell cytosol. Both molecular biologic and molecular genetic methodologies, which include recombinant DNA, site-directed mutagenesis, DNA sequence analysis, as well as protein characterization and in vitro toxicity determinations will be used during the course of this investigation. The long-range goals of this investigation are to understand the molecular mechanisms involved in the iron-mediated regulation of diphtheria tox expression, and the comparative analysis of tox promoter activity in C. diphtheriae and E. coli. We will also provide a detailed understanding of diphtheria toxin structural/functional relationships by site-directed point and deletion mutagenesis and subsequent characterization of mutant toxin-related polypeptides.